leukocyte function defect

1. LEUKOCYTE FUNCTIONS
DEFECTS
Dr Nidhi Rai

2. Introduction
• Essential cellular components of blood.
• Develop from the pluripotential hematopoietic stem
cells in the bone marrow.
• Leukocytes include
 Granulocytes:
– Neutrophils
– Basophils
– Eosinophils
 Monocyte
 Lymphocyte

3. Primary function: To protect the host from
infectious agents or pathogens.
Innate Immune Response
Acquired/Adaptive /Specific
immune system
• 1st response to
common class of
pathogens.
• Neutrophils &
Monocytes – Play
a major role.
• Rapid but limited.
• Initiated in
lymphoid tissue
when pathogens
encounter
lymphocytes.
• Lymphocytes –
plays a major role.

4. Neutrophils
• Neutrophils are very mobile cells
• Two types of granules:
– Primary or azurophilic
– Secondary or specific
• All granules contain enzymes which are involved in killing
and digesting bacteria and fungi.

5. Functions of Neutrophils
• Innate immune response: 4 steps
1. Adherence
2. Migration ( chemotaxis)
3. Phagocytosis
4. Bacterial killing

6. 1. Adherence
• Activation of leukocytes & vascular endothelial cells (VEC)
by inflammatory mediators(cytokines).
• Activation result is activity of 3 classes of cytoadhesion
mol. (CAM) & their ligands.
• CAM & their ligands play a major role.
1. Selectins & their ligands
2. Intercellular adhesion molecule (ICAM)
3. Beta 2 (CD18) family of leukocyte integrin & their
ligands.

8. 2. Migration- pseudopodia forms
ICAM
E & P Selectins

9. 3. Phagocytosis
Some pathogens are
recognized by opsonization
(IgG & C3b)
PAMP
PRR
Degranulation

10. 4.Bacterial Killing & Digestion
• Phagosomes  fuse with the lysosome( phagolysosome)
 discharge their granules  Bacterial killing &
digestion.
• Microbicidal mechanism – 2 types
1. Oxygen dependent
2. Oxygen independent

11. Oxygen Dependent
• Neutrophils activation is accompanied by increase in O2
use- Respiratory burst/ oxidative burst.
• Respiratory burst – caused by activated NADPH oxidase.
 Increase in O2 consumption.
 Increase glycolysis.
 Production of ROS ( superoxide)
 Increase in glucose oxidation by HMP shunt
• Activated NADPH – detected by NBT, Cytochrome oxidase
or chemiluminescence test.

12. • MPO independent:
 H2O2
 Superoxide ion
 Hydroxyl ion
 Singlet oxygen
• MPO dependent: Present in primary granules

13. Oxygen Independent
• Mediated by the granules present in primary,gelatinase,specific
granules.
• Acid pH of phagosomes  Increase activity of granular protein
 Microbicidal activity
• Alone insufficient to kill microbes.
• Include :
 Lysozyme
 Lactoferrin
 BPI( Bacterial permeability inducing proteins)
 Defensins
 Collagenase

14. Monocytes Functions
• Active in both innate and adaptive IR.
• Phagocytosis.
• Ingest activated clotting factors thus limiting the
coagulation process.
• Secrete a variety of substance affects the functions of
other cells especially lymphocytes.

15. Eosinophils
• Eosinophils : 2 - 8 % leukocytes
• Derived from hematopoietic stem cells
• bilobed nucleus with numerous bright orange
cytoplasmic granules
• Originates from IL -5 responsive CD 34 + myeloid
progenitor cells
• IL -5 has lineage specificity for eosinophils

16. Function of Eosinophils
• Cationic proteins, cytokines and leukotriens- in
eosinophil granules mediate
 Parasite defense
 Allergic response
 Tissue infiltration
 Immune modulation

17. Basophils
• Basophils are least of the circulating leucocytes
• They constitute less than 1% of the circulating leucocytes
or absolute counts of 0.02-0.08x109/lt
• Normal range on BMA is 0-0.5%
• Circulates in blood and normally not found in tissues
• Recruited to tissues during inflammatory or immunologic
response

18. Functions of Basophils
• Basophils – Key player in Allergy
• Degranulation
• Late Phase Reaction

19. Lymphocytes
• Specific component of immune system.
• Different types :
 B-Lymphocytes (“B cells)
 T- Lymphocytes (“T cells”)
Natural Killer Cells (NK cells)
Memory Cells
Suppressor Cells
• They have different functions in specific immunity.

20. B-LYMPHOCYTES
• Mature in bone marrow, then carried to lymphoid tissue
via blood stream and lymphatic circulation.
• This process of maturation and migration takes place
throughout life.
• Other lymphocytes can be generated via mitosis of B
lymphocytes resident in lymphoid tissues

21. T-LYMPHOCYTES
• Immature lymphocytes leave bone marrow during fetal
and early neonatal life.
• Go to thymus gland.
• Mature there before they go on to other lymphoid
tissues.

22. Role in Specific Immunity
• The body must be able to recognize the difference
between “self” and “nonself.”
• Any lymphocytes with antibodies that recognize one’s
own body tissue as an antigen are killed during fetal life.
• Stages of specific immune response-
1) Antigen encounter and recognition by lymphocytes.
2) Lymphocyte activation.
3) Attack

23. 1) Antigen encounter and recognition by lymphocytes
• Specific lymphocytes are programmed to recognize a
specific antigen.
• Usually happens in a lymphoid organ, bloodstream, or
lymph vessel.
2) Lymphocyte activation:
• Once a lymphocyte has recognized an antigen, it
undergoes numerous cycles of mitotic divisions, making
more of the same.
• Some of the newly produced cells carry out the attack;
others influence the activation and function of the attack
cells

24. 3) Attack:
B-lymphocytes have specific receptors on their cell
membrane – ANTIBODIES – that bind with invading
materials/organisms

25. Function of T Lymphocytes
• Do not produce antibodies.
• Function in “cell-mediated immunity.”
• “NATURAL KILLER” cells destroy viruses.
• Secrete “lymphokines” which attract phagocytic cells.
• Secrete “perforin” which eats the cells membrane or
viral coat of invaders.

26. • Helper T cells:
• Induce macrophages to destroy other antigens
• STIMULATE B-LYMPHOCYTES TO PRODUCE
ANTIBODIES.
• “Suppressor T Cells” prevent overreaction of the system.
(Inhibit B-lymphocye production.)

27. Disorders of Leukocyte Function
1. Disorders of opsonisation and ingestion
2. Defective adhesion of Leukocytes.
a) Leukocyte adhesion deficiency
b) Drug induced
3. Defective locomotion and chemotaxis
a) Actin polymerization abnormalities.
b) Neonatal neutrophils
c) Interleukin 2 administration
d) Cardiopulmonary bypass
e) Enhanced motile response

28. 4. Defective microbial killing
a) Chronic granulomatous disease
b) RAC – 2 deficiency
c) Myeloperoxidase deficiency
d) Hyper immunoglobulin syndrome
e) G6PD deficiency
f) Extensive burns
g) Glycogen storage disease
h) Ethanol toxicity
i) End stage renal disease
j) Diabetes mellitus

29. 5. Abnormal structure of the nucleus or of an organelle
a) Hereditary macropolycytes
b) Hereditary hyper segmentation
c) Pelger- Huet anomaly
d) Alder- Reilly anomaly
e) May – Hegglin anomaly
6. Degranulation defect
a) Chediak – Higashi disease
b) Specific granule deficiency

30. Disorders of opsonisation and ingestion
Basic Pathogenesis Defect Impaired
Function
Clinical
consequence
• Synergistic action of
Ig & complement
creates opsonins
•Coats microorganism
• Stimulate
chemotaxis
•Def  impaired Fn
• Ig & C3
deficiencies
• Properdin
deficiency
• Mannose
binding
protein def.
Deficiency
of
chemotaxis
& opsonins
activity
Recurrent
pyogenic
infections

31. Defective Adhesion
Main functional defect
• Unable to successfully activate and "grab" or adhere to
the blood vessel wall because they lack the necessary
podium & if present but may be defective and not work
properly.
• Since these leukocytes cannot adhere, experience an
"adhesion deficiency",

32. Leukocyte Adhesion deficiency
Dis
order
Inherit
ance
Defect Genetic
defect
Impaired
Function
Clinical
course
LAD – 1 AR Absence of CD11/CD18
surface adhesive GP( B
integrin) on leukocyte
membrane
Failure to
express CD
18 m RNA
• Rolling
normal
•Decreased
binding of C3bi
to leukocytes
•Impaired
adhesion to
ICAM 1 & 2.
• Neutrophilia
• Recurrent
bacterial
infection.
• Delayed
umbilical
cord
separation
LAD – 2 AR Loss of fucosylation of
ligands for selectin ( Cd15s
expression)
Mutation
of the GDP
fucose
transporter
• Donot roll
well.
•Decreased
adhesion
• Neutrophilia
• Recurrent
bacterial
infection
without pus.
LAD – 3 AR Impaired integrin
functions.
Defective kindlin – 3 ,
binds to B- integrin ->
integrin activation
Mutation
of FERMT
3- encodes
kindlin- 3
• Impaired
leukocyte
adhesion &
platelet
activation
• Neutropenia
• Recurrent
infections.
• Bleeding
tendency

33. Disorder of Cell Motility
Disorder Inherit
ance
Defect Genetic
defect
Impaired
function
Clinical
course
1.Enhanced
motile response
( familial
Mediterranean
fever)
AR Defective Pyrin
regulation of
caspase 1 &
thereby IL-B
secretion.
Mutation a
gene encodes
pyrin(
chromosome
– 16)
• Increase
sensitivity to
endotoxin.
• Excessive
accumulation
of leukocytes.
• Recurrent
fever.
•Peritonitis.
•Pleuritis.
• Arthritis
• Amyloidosis
2.Immue
complexes
• Rheumatoid
arthritis.
• SLE & other
inflammatory
condition.
•Binds to Fc
receptors on
neutrophils.
• Impaired
chemotaxis
Recurrent
pyogenic
infections
3. Actin
polymerization
abnormality
• Actin doesn’t
polymerize well.
• Lower level of
F-actin.
Over
expression of
Leufactin (
lymphocyte
specific
protein 1)- F
actin binding
Defect in
chemotaxis,
adhesion and
phagocytosis
Recurrent
bacterial
infections

34. Defective microbial killing
• The ultimate step in the elimination of infectious agents
and necrotic cells is their killing and degradation within
neutrophils and macrophages.
• Any defect or deficiency causes recurrent infections

35. Chronic Granulomatous Disease
• Affecting the functions of neutrophils & monocytes.
• Defect : Absent or Reduced function of the respiratory
burst.
• Genetic defect: Congenital defects in the five components
of the enzyme NADPH oxidase.
• Clinical features: Rucurrent catalase +ve infections
• Hallmark : presence of granulomas caused by thechronic
inflammatory response to the pathogen

36. • Functional defects: Defects in opsonisation, phagocytosis,
or intracellular killing.
• Lab diagnosis:
1. Bacterial killing test
2. Phorbol myristate acetate (PMA)
3. Nitroblue tetrazolium (NBT) test
4. Flow cytometric analysis (Most sensitive)
5. Prenatal diagnosis of CGD :Analysis of DNA from
chorionic villous sampling or amniotic fluid cells

37. Defective Microbial Killing
Disorder Inheri
tance
Defect Genetic
defect
Impaired
functions
Clinical
course
1. G6PD
Deficiency
< 5 % of normal
activity of G6PD
• Failure to
activate NADPH
oxidase( required
for phagocytosis).
• Hemolytic
anemia
Infection with
catalase +ve
organism
2. RAC – 2
Deficiency
AD Negative
inhibition by
mutated protein
of Rac- 2
mediated
function
Mutation in
Rac- 2
Failure to activate
receptor mediated
O2 generation &
chemotaxis.
• Neutrophilia.
• Recurrent
bacterial
infections
3. Deficiencies
of glutathione
reductase &
synthetase
AR Due to def.
Failure to
detoxify H2O2
Excessive
formation of
H2O2
Minimal
problem with
recurrent
pyogenic
infections

38. Defective Microbial Killing
Disorder Inherit
ance
Defect Genetic
defect
Impaired
functions
Clinical course
4. Hyperimmuno
globulin – E
syndrome ( Job
syndrome)
• AD
• Spora
dic
• Defective
STAT 3
protein
( major
transductio
n protein)
Missense
mutation or
in-frame
deletion in
STAT 3
protein
• Poor Ab &
cell mediated
responses.
• Impaired
chemotaxis.
•Impaired
regulation of
cytokine
productions.
• Recurrent skin
& sinopulmonary
infection
• Retained Pri
teeth
• Facies
characteristic
• Blood &
sputum
eosinophilia.
5. MPO deficiency AR Absence of
MPO In N.
& M
Failure to
process
modified
precursor
protein due
to missense
mutation
H2O2
dependent
antimicrobial
activity not
potentiated by
MPO.

39. Degranulation Abnormalities
• Normally in stimulated cells signal transduction cascade
activates G proteins enhanced intracellular Ca2+ ,
protein kinase activation.
• Culminate in secretion ( Fusion of granule membrane
with phagosomes of the plasma membrane) –
Degranulation.
• Defect in degranulation- Reduce bactericidal activity
( Defective Leukocytic function)

40. Chediak- Higashi Syndrome
• Congenital gigantism of peroxidase positive granules.
• Inheritance – AR
• Basic defect : Increased fusion of cytoplasmic granules 
Abnormal large peroxidase positive granules.
• Giant granules are formed in most granules containing cells
through out the body.
• Genetic defect: CHS1/LYST gene –encodes a protein , regulate
granule fusion.

41. Pathogenesis:
Early stage of Myelopoiesis
Azurophilic granules fuse to form
giant granules
Large granules- Reduced hydrolytic
enzymes
Precursors -
phagocytosed in the
marrow
Moderate Neutropenia
In PB- Normal ingestion of
particles & O2 met. By
Leukocyte
Slow & inconsistence delivery of
hydrolytic enzymes
Slow Microbial killing
Bacterial
Infection
CHS Cell Mb.-
More fluid than
normal.
1. Fusion of
granules.
2. Reducing
expression of
CD11b/CD18(M
ac-1)
3. Increase C-AMP
Decrease
chemotaxis

42. • Monocytes – same functional derangement.
• Perforin deficient NK cells impair cytotoxic activity 
unable to kill microbes.

44. Clinical Course:
• Characteristically present with partial albinism , silvery
hair & photophobia.
• Recurrent pyogenic infections.
• Increase bleeding tendencies.
• Usually quiescent.
• >85% of patients Accelerated phase
 Lymphadenopathy, neutropenia,hepatosplenomegaly as
a manifestation of hemophagocytic syndrome.

45. Lab Findings:
• Characteristic microscopic findings
1. Large, often multiple, peroxidase +ve granules in the
granulocyte of blood and BM.
2. Large melanosomes in the hair.
• Abnormal platelet aggregation (def. of storage pool of
ADP & serotonin).
• Early phase- blood counts normal
• Late stage – cytopenias.

46. Specific Granules Deficiency
Inheritance Functional
defect
Genetic defect Impaired
Function
Lab . Findings
• AR
• Acquired
form
 Thermally
injured Pt.
 MDS
• Absence of
specific granules
(ALP+)
• MPO +ve
primary granules
present.
• Confined to
myeloid cells
only.
• Functional loss
of myeloid
transcription
factor due to
Mutation or
reduced
expression of
growth factor
independence
(Gfi) and C/EBPE
which regulates
specific granules
formation
• Impaired
chemotaxis &
bactericidal
activity.
• Bilobed nuclei
in neutrophils.
•Def. of
Defensin,gelatina
se, collgenase, vit
B12 binding
protein &
lectoferrin.
(Present in sec
granules.)
• Presence of
neutrophils
devoid of specific
granules but
containing
azurophilic
granules on PB.
• Confirmatory
test- def. Of
lactoferrin or vit
B12 binding
protein.

47. Abnormal Structure of the Nucleus or
Organelle
Disorder Morphological defect Impaired
function
Clinical course or
associated condition
1. Pelger –
Huet
Anomaly
• Distinct shapes of the nuclei of
leukocyte with decrease number of
nuclear segment.
• Nuclei –Rod like , dumb bell shaped,
pinch nez with small, round or oval
individual lobes.
Function
normally
2. Alder –
Reilly
Anomaly
• Large, dark azurophilic & basophilic
granules in all leukocytes.
•Distinguish from toxic granules by
staining metachrmoatically with
toluidine blue.
• Lymphocytes – occur in clusters
surrounded by vacuoles & shaped like
a dot or comma ( Gasser’s cell)
Associated with
Mucopolysaccharidosis

48. Abnormal Structure of the Nucleus or
Organelle
Disorder Morphological Defect Impaired
Functions
Clinical course /
associated
conditions
3. May Hegglin
Anomaly
( Autosomal
dominant)
•Giant fused , well defined
granules in granulocytes &
lymphocytes.
• Inclusion similar to Dohle
bodies – distinguished from
it as these are more large &
rounded.
Engulf but
donot kill
microorganism.
• Serious, often fatal.
• Repeated pyogenic
infections
4. Hereditary
Hypersegmentation
( Autosomal
dominant)
Increase number of
neutrophilc segments
5. Hereditary
hypersegmentation of
eosinophils and –ve
staining for
peroxidase
Lack of sudanophilia &
peroxidase activity

49. Basophilic inclusions
May Hegglin anomaly
Note the granules
Alder Reilly anomaly-
hurler’s syndrome
Pelger Huet Anomaly

50. Workup of the Patient with
Recurrent Infections

51. 1..Initial evaluation
• History, physical
examination.
• Family history
• Blood counts
•Bacterial counts
Functional asplenia
Wiskot- Aldrich syndrome
Neutropenia work up
( BM,ANA,CBC)
Howell Jolly Bodies
Thrombocytopenia,
eczema
Neutrophil <1500
Neutrophil G6PD def.
Chediak – Higashi
syndrome
Specific granules Def.
Hemolytic anemia
Partial albinism,
abnormal granules
Abnormal granules
Pelger-Huet anomaly
If normal
•2. Ig/Complement
work up- Ig Levels.
•Ab titers
• Delayed
hypersensitivity
•Total T cells
Hypogammaglobulin
syndromes
e.g. linked
agammaglobulinemia
Severe combined immuno
def, AIDS, Mucocutaneous
candidiasis,ataxia-
telegiectasia
Hypocomplementemia
syndrome
IgE.- 2000 IU/ml
Hyper- IgE
Decreased Ig
Cellular Immuno
def.
Decreased complement
Stat 3 mutation
If normal
3. Phagocyte
evaluation.
•NBT
•DHR assay
• Chemotaxis assay
If normal
Neutrophil G6PD def.
LAD.Chediak-Higashi
syndrome, Specific granules
Def,Rac-2 def.
Chronic granulomatous ds
Complement def, humoral
defects
Absent O2-,
Abnormal NBT,DHR
Only abnormal chemotaxis
Abbreviated O2- production
- GSH pathway
Abnormal response to
activated control serum
4.Further phagocyte
evaluation.
• MPO stain
•Flow
cytometry(CD11/18)
•Quantitative
ingestion assay
MPO Def
Opsonins Defect
Neutrophil actin dysfn
MPO absent
Decrease ingestion
with Pt’s serum
Decreases ingestion
with control serum
LAD-1.
LAD-2
LAD-3.
Absent Cd11/18
Decreased sLe
expression by flow
Failure to generate O2-
when challenged with
unopsonized zymosam

52. References
• Wintrobes clinical hematology
• William’s hematology
• Postgraduate hematology
• McKenzie Clinical lab hematology
• Robbins basic pathology

53. Thank You